Pre-mRNA splicing is a fundamental process required for the expression of most metazoan genes. Defects in splicing lead to many human genetic diseases, and splicing mutations in a number of genes involved in growth control have been implicated in multiple types of cancer. Insights into the basic mechanisms of pre-mRNA splicing and splice site recognition are therefore fundamental to understanding regulated gene expression and human disease. While many different cis-acting RNA splicing elements have been shown to influence alternative splicing, it is currently unknown how their combinatorial contribution mediates exon inclusion or exclusion. Complicating the task of experimentally deciphering alternative splicing decisions is the fact that most human genes contain multiple introns and exons that often exhibit more complex splicing patterns than simply selecting between two competing splice sites. This renewal application focuses on understanding the mechanisms of regulated splice-site selection with the long-term goal to predict alternative splicing based on sequence analysis. The experiments outlined below build on the most exciting discoveries made during the previous funding period. Historically, SR proteins have been associated with splicing activation, whereas hnRNPs are known for their inhibition of the splicing reaction. However, new genome-wide analyses suggested that hnRNP-like splicing factors could also activate exon inclusion. We demonstrated that both classes of splicing regulators have the ability to promote or repress splicing, antagonistic activities that simply depend on whether the splicing regulator binds within the exon or within the intron. Thus, SR protein and hnRNPs are functionally interchangeable and their regulation of splicing is dependent on the location of their binding site relative to a splice site. How is it possible that a splicing factor can activate or repress spliceosome assembly? We propose to carry out complementing sets of experiments to determine the molecular mechanisms that switch splicing regulatory proteins from splicing activators to splicing repressors (Specific Aims 1 and 2). In Specific Aim 3 we will determine the frequency position-dependent splicing in living cells and use the new molecular insights to improve splicing predictions.